Development of a Metastatic Breast Cancer model in the nude rat for MRI Cell Tra

本研究的目的是利用核磁共振(MRI)扫描仪和体内生物发光成像(BLI)的多模式成像方法，建立大鼠乳腺癌脑转移瘤模型，以监测疾病的自然病程。MDA-MB231BR细胞系是一种脑转移乳腺癌细胞系，稳定表达萤火虫荧光素酶基因(Luc)，并在体外用超顺磁性氧化铁纳米颗粒(Spion)进行磁性标记，用于早期MRI监测。裸鼠心脏内注入标记MDAMB231BRLUC(231BRL)细胞的Spion Ferumoxide。FEPro标记的~(231)BRL细胞注射后，大鼠出现多发性脑和脊髓转移，所有脑部病变的大鼠均出现多发性骨转移。心内注射Spion标记细胞后1周，动物体内可检测到普鲁士蓝阳性乳腺癌细胞。BLI显示大鼠脑和骨骼中的荧光素酶光子流活性增加，而MRI显示在注射细胞后的前3天内，脑内有许多与Spion标记细胞相对应的低信号区域。量化靶组织中标记干细胞的数量对于优化细胞治疗的剂量和时机具有重要意义。 我们使用多梯度回波脉冲序列来计算T2*弛豫时间，以改进体素内标记细胞的检测。我们还研究了血氧水平依赖(BOLD)MRI用于评估转移性肿瘤的氧合和血流灌注。目前还不清楚BOLD除了具有预测预后的潜力外，是否还可以用于诊断。注射FEPro乳腺癌细胞后第1天，在大鼠脑部的T2*W图像上可检测到许多低信号区域，与血管内的肿瘤细胞簇一致。在注射细胞后第36天，体外T2加权磁共振图像上共发现34个皮质肿瘤。这些区域被映射到活体图像上。在这些肿瘤中，26个肿瘤的相应区域与前一周(第29天)MRI相比有明显的大胆下降，17个肿瘤在2周前(第22天)MRI有明显的大胆下降。更重要的是，当肿瘤直径小于500m时，BOLD效应就可以被检测到。BOLD MRI是肿瘤转移的早期标志，其变化与未诊断的微转移疾病的血管异常相一致。尽管很难将大胆的变化归因于氧合或血流灌注，但BOLD MRI可能有助于发现正在发展的转移瘤。 治疗乳腺癌脑转移的药理学方法收效甚微。在一定程度上，血脑屏障(BBB)的不通透性阻碍了化疗药物向脑内转移瘤的输送。血脑屏障渗透性化疗药物正在开发中，非侵入性评估这些药物的疗效在临床前和临床环境中都将是重要的。我们使用动态对比增强(DCE)和扩散加权成像(DWI)磁共振成像(MRI)技术来监测大鼠脑内转移性乳腺癌、肿瘤血管通透性和细胞密度。我们证明，在MB 231BR模型中，脑转移和骨转移具有明显的生理特征，如MRI所测量的那样。脑转移瘤与周围脑相比，DCE评估的血脑屏障通透性有限，DWI测量的表观扩散系数(ADC)增加。显微镜下，我们发现脑转移瘤高度浸润性，通过血管选择性生长，并对周围神经元及其树突造成广泛的水肿和损伤。通过比较，位于软脑膜或骨骼的转移瘤具有高的血管通透性和显著的低ADC值，提示细胞增多。组织学检查，骨和软脑膜肿瘤为实性肿块，肿瘤边缘清楚。这些组织部位的不同特点突出了微环境对转移瘤生长的影响。这项工作提出了一个问题，即DWI和DCE是否适合于评估化疗和抗血管生成药物用于治疗选择性脑转移瘤的疗效，而这些技术则用于实体瘤肿块。

The purpose of this study was to develop a metastatic brain tumor model of breast cancer in the rat to monitor the natural history of the disease using a multimodal imaging approach with magnetic resonance imaging (MRI) scanner and in vivo bioluminescence imaging (BLI). MDA-MB231BR cell line is a brain seeking metastatic breast cancer line that was stably transfected to express the firefly luciferase cDNA (LUC) and was magnetically labeled with superparamagnetic iron oxide nanoparticles (SPION) ex vivo for early monitoring by MRI. Nude rats underwent intracardiac infusion SPION ferumoxides labeled MDAMB231BRLUC (231BRL) cells. FEPro labeled 231BRL cells injected rats developed multiple brain and spinal cord metastasis and all rats with brain lesion had multiple skeletal metastasis. Prussian blue positive breast cancer cells could be detected in animals up to 1 week following intracardiac injection of SPION labeled cells. BLI demonstrated increase in luciferase photon flux activity in the brain and bones of the rats while MRI revealed numerous hypointense regions corresponding to SPION labeled cells in the brain within the first 3 days following injection of cell. Quantifying the number of labeled stem cells in target tissues is of great importance to optimize dose and timing of cellular therapy. We has employed a multi-gradient echo pulse sequence to calculate T2* relaxation times to improve the detection of labeled cells within the voxel. We also have investigated the use of Blood oxygen level dependant (BOLD) MRI has been used to evaluate metastatic tumor oxygenation and perfusion. It is unknown whether BOLD can be used for diagnosis in addition to its prognostic potential. Day 1 post injection of FEPro breast cancer cells, numerous hypointense regions were detected on T2*w images throughout rat brain consistent with clusters of tumor cells in the vasculature. At day 36 post injection of cells a total of 34 cortical tumors were identified on the T2-weighted ex vivo MR images. These regions were mapped to the in vivo images. Of these tumors, 26 had noticeable BOLD decreases in the corresponding region from the week prior (day 29) MRI, and 17 had BOLD decreases 2 weeks prior (day 22) MRI. Importantly, the BOLD effects could be detected when the tumor was less than 500 m. BOLD MRI was an early marker of metastatic tumor development, and the changes are consistent with vascular abnormalities from undiagnosed micrometastasis disease. Despite the difficulties in attributing BOLD changes to oxygenation or perfusion, BOLD MRI may aid the detection of developing metastases. Pharmacological approaches to treat breast cancer metastases in the brain have been met with limited success. In part, the impermeability of the blood brain barrier (BBB) has hindered delivery of chemotherapeutic agents to metastatic tumors in the brain. BBB-permeable chemotherapeutic drugs are being developed, and noninvasively assessing the efficacy of these agents will be important in both preclinical and clinical settings. We used dynamic contrast enhanced (DCE) and diffusion weighted imaging (DWI) are magnetic resonance imaging (MRI) techniques to monitor metastatic breast cancer in the rat brain, tumor vascular permeability and cellularity. We demonstrate that brain and bone metastases in the MB 231BR model develop with distinct physiological characteristics as measured with MRI. Brain metastases had limited permeability of the BBB as assessed with DCE and an increased apparent diffusion coefficient (ADC) measured with DWI compared to the surrounding brain. Microscopically, we showed that brain metastases were highly infiltrative, grew through vessel co-option, and caused extensive edema and injury to the surrounding neurons and their dendrites. By comparison, metastases situated in the leptomenengies or in the bone were demonstrated to have high vascular permeability and significantly lower ADC values suggestive of hypercellularity. On histological examination, tumors in the bone and leptomenengies were solid masses with distinct tumor margins. The different characteristics of these tissue sites highlighted the influence of the microenvironment on metastatic tumor growth. The implication of this work raises the issue of the suitability of DWI and DCE to evaluate the response of chemotherapeutic and anti-angiogenic agents used to treat co-opted brain metastases in contrast to these techniques being used in solid tumor masses